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Abstract:

Industrial-scale fermentative production of 3-carboxy-cis,cis-muconic acid
from terephthalic acid. Also, a protocatechuate 4,5-ring-cleaving enzyme
gene-disrupted strain in which the gene coding for (a) the amino acid
sequence set forth in SEQ ID NO: 1 or 3, or (b) the amino acid sequence
set forth in SEQ ID NO: 1 or 3 which has a deletion, substitution,
addition and/or insertion of one or more amino acids and exhibits
protocatechuate 4,5-ring cleavage activity, present in the chromosomal
DNA of microbial cells, has been disrupted; recombinant plasmids
comprising the Tph gene and protocatechuate 3,4-dioxygenase gene;
transformants obtained by introducing the recombinant plasmids into the
disrupted strain; and a process for production of
3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone
characterized by culturing the transformants in the presence of
terephthalic acid.

Claims:

1. A protocatechuate 4,5-ring-cleaving enzyme gene-disrupted strain in
which the gene coding for:(a) the amino acid sequence set forth in SEQ ID
NO: 1 or 3, or(b) the amino acid sequence set forth in SEQ ID NO: 1 or 3
which has a deletion, substitution, addition and/or insertion of one or
more amino acids and exhibits protocatechuate 4,5-ring cleavage
activity,present in the chromosomal DNA of microbial cells, has been
disrupted.

2. A protocatechuate 4,5-ring-cleaving enzyme gene-disrupted strain in
which a gene that:(a) comprises the nucleotide sequence set forth in SEQ
ID NO: 2 or 4; or(b) is a nucleotide sequence hybridizing with DNA
consisting of a nucleotide sequence complementary to the nucleotide
sequence of (a), under stringent conditions, and coding for an enzyme
with protocatechuate 4,5-ring cleavage activity,present in the
chromosomal DNA of microbial cells, has been disrupted.

3. A gene-disrupted strain according to claim 1 or 2, in which the
protocatechuate 4,5-ring-cleaving enzyme gene has been disrupted by
homologous recombination between a gene coding for:(a) the amino acid
sequence set forth in SEQ ID NO: 1 or 3, or(b) the amino acid sequence
set forth in SEQ ID NO: 1 or 3 which has a deletion, substitution,
addition and/or insertion of one or more amino acids and exhibits
protocatechuate 4,5-ring cleavage activity,present in the chromosomal DNA
of microbial cells, and homologous recombination DNA having a DNA
sequence that can undergo homologous recombination with the gene and
lacking protocatechuate 4,5-ring cleavage activity.

4. A gene-disrupted strain according to any one of claims 1 to 3, wherein
the parent strain of the protocatechuate 4,5-ring-cleaving enzyme
gene-disrupted strain is a Comamonas sp. bacterium.

7. A transformant obtained by introducing a recombinant plasmid according
to claim 6 into a gene-disrupted strain according to any one of claims 1
to 5.

8. A process for production of 3-carboxy-cis,cis-muconic acid and/or
3-carboxymuconolactone, characterized by culturing a transformant
according to claim 7 in the presence of terephthalic acid.

Description:

TECHNICAL FIELD

[0001]The present invention relates to a protocatechuate 4,5-ring-cleaving
enzyme gene-disrupted strain, to recombinant plasmids comprising a gene
coding for an enzyme participating in a multistage reaction process for
fermentative production of 3-carboxy-cis,cis-muconic acid and/or
3-carboxymuconolactone from terephthalic acid via protocatechuic acid, to
transformants incorporating the recombinant plasmid in the disrupted
strain, and to process for industrial production of
3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone using the
same.

BACKGROUND ART

[0002]Terephthalic acid is an aromatic compound separated from petroleum
components, and it is cheaply mass-produced as a starting material for
PET. Development of new biodegradable functional plastics using
terephthalic acid as the starting material will allow copolymerization
with petroleum-based polymer materials such as PET, to permit the
development of polymer materials with excellent biodegradability.

[0003]The present inventors have found that the terephthalic
acid-degrading microorganism, Comamonas sp. E6, can completely degrade
terephthalic acid via 2H-pyran-2-one-4,6-dicarboxylic acid after first
converting it to protocatechuic acid (Patent document 1). There have also
been reported a recombinant vector comprising the genes coding for
terephthalate dioxygenase (TPA-DOX),
1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase
(DCD-dehydrogenase), terephthalate transporter (TPA transporter) and a
positive regulator, by removing the genes coding for protocatechuate
4,5-dioxygenase and 4-carboxy-2-hydroxy-6-semialdehyde muconate
dehydrogenase from the chromosomal DNA of the microorganism,
transformants containing the vector, and a method of producing
2H-pyran-2-one-4,6-dicarboxylic acid from terephthalic acid using the
transformants (Japanese Patent Application No. 2005-298242).

[0005]In the process, terephthalic acid is converted to 3-carboxy-cis and
cis-muconic acid via 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate
and further via protocatechuic acid, and the 2,3-bond, 3,4-bond or
4,5-bond of protocatechuic acid is cleaved, depending on the type of
microorganism (hereunder, 2,3-bond cleavage, 3,4-bond cleavage and
4,5-bond cleavage of protocatechuic acid will be referred to as "2,3-ring
cleavage", "3,4-ring cleavage" and "4,5-ring cleavage", respectively). In
the presence of certain microorganisms, the 3-carboxy-cis,cis-muconic
acid as the 3,4-ring cleavage product of protocatechuic acid is further
catabolized via 3-carboxymuconolactone or 4-carboxymuconolactone.

[0006]No process has been known to date for fermentative production of
3-carboxy-cis,cis-muconic acid using terephthalic acid as the starting
material.

DISCLOSURE OF THE INVENTION

[0007]It is an object of the present invention to provide a process for
industrial-scale fermentative production of 3-carboxy-cis,cis-muconic
acid from terephthalic acid via protocatechuic acid, and/or a process for
fermentative production of 3-carboxy-cis,cis-muconic acid from
terephthalic acid via protocatechuic acid and acid treatment thereof to
obtain 3-carboxymuconolactone on an industrial scale.

[0008]In order to obtain 3-carboxy-cis,cis-muconic acid efficiently, it is
necessary to disrupt genes having 2,3-ring cleavage function, 3,4-ring
cleavage function or 4,5-ring cleavage function, or to disrupt genes that
further metabolize 3-carboxy-cis,cis-muconic acid.

[0009]The present inventors have conducted ardent research on this
subject, and as a result have considered that disrupting the cleavage
activity of protocatechuate 4,5-ring-cleaving enzyme would completely
disrupt the conversion process from protocatechuic acid to
2H-pyran-2-one-4,6-dicarboxylic acid, and have therefore generated
protocatechuate 4,5-ring-cleaving enzyme gene-disrupted strains in which
the cleavage activity of protocatechuate 4,5-ring-cleaving enzyme has
been disrupted. It was further found that it is possible, using the
disrupted strains, to produce 3-carboxy-cis,cis-muconic acid and/or its
acid treatment product, 3-carboxymuconolactone, from terephthalic acid at
high yield and inexpensively.

(a) the amino acid sequence set forth in SEQ ID NO: 1 or 3, or(b) the
amino acid sequence set forth in SEQ ID NO: 1 or 3 which has a deletion,
substitution, addition and/or insertion of one or more amino acids and
exhibits protocatechuate 4,5-ring cleavage activity, present in the
chromosomal DNA of microbial cells, has been disrupted.(2) The present
invention further provides a protocatechuate 4,5-ring-cleaving enzyme
gene-disrupted strain in which a gene that comprises:(a) the nucleotide
sequence set forth in SEQ ID NO: 2 or 4; or(b) a nucleotide sequence
hybridizing with DNA consisting of a nucleotide sequence complementary to
the nucleotide sequence of (a), under stringent conditions, and coding
for an enzyme with protocatechuate 4,5-ring cleavage activity,present in
the chromosomal DNA of microbial cells, has been disrupted.(3) The
invention further provides a gene-disrupted strain according to (1) or
(2), in which the protocatechuate 4,5-ring-cleaving enzyme gene has been
disrupted by homologous recombination between a gene coding for:(a) the
amino acid sequence set forth in SEQ ID NO: 1 or 3, or(b) the amino acid
sequence set forth in SEQ ID NO: 1 or 3 which has a deletion,
substitution, addition and/or insertion of one or more amino acids and
exhibits protocatechuate 4,5-ring cleavage activity, present in the
chromosomal DNA of microbial cells, and homologous recombination DNA
having a DNA sequence that can undergo homologous recombination with the
gene and lacking protocatechuate 4,5-ring cleavage activity.(4) The
invention further provides a gene-disrupted strain according to any one
of (1) --(3), wherein the parent strain of the protocatechuate
4,5-ring-cleaving enzyme gene-disrupted strain is a Comamonas sp.
bacterium.(5) The invention further provides a gene-disrupted strain
according to (4), wherein the Comamonas bacterium is Comamonas sp. E6.(6)
The invention further provides a recombinant plasmid comprising a
terephthalate dioxygenase gene (TPA-DOX gene), NADPH-reductase gene,
1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase gene
(DCD dehydrogenase gene), positive regulator gene, terephthalate
transporter gene (TPA transporter gene) and protocatechuate
3,4-dioxygenase gene (pcaHG gene).(7) The invention further provides a
transformant obtained by introducing a recombinant plasmid according to
(6) into a gene-disrupted strain according to any one of (1)-(5).(8) The
invention further provides a process for production of
3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone,
characterized by culturing a transformant according to (7) in the
presence of terephthalic acid.

[0011]According to the invention it is possible to accomplish high-yield
and inexpensive fermentative production of 3-carboxy-cis,cis-muconic acid
and/or 3-carboxymuconolactone from terephthalic acid.

[0016]FIG. 5 shows disruption of the pmdB1 gene in Comamonas sp. E6. 5(A)
is an illustration of a method of constructing an E6 pmdB1 gene-disrupted
strain, and 5(B) shows the results of Southern hybridization analysis of
the pmdB1 gene-disrupted strain.

[0017]FIG. 6 is an illustration showing construction of the recombinant
plasmid pKHG.

[0018]FIG. 7 is an illustration showing construction of the recombinant
plasmid pKHG/C.

[0019]FIG. 8 is an illustration showing construction of the recombinant
plasmid pKTphHG/C.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020]The gene-disrupted strain of the invention may be one in which at
least one function of the 2,3-ring cleavage function, 3,4-ring cleavage
function and 4,5-ring cleavage function is disrupted. Alternatively, the
gene-disrupted strain of the invention may retain the 3,4-ring cleavage
function, in which case the function of further metabolizing
3-carboxy-cis,cis-muconic acid must be disrupted. In order to disrupt the
activity of metabolizing carboxy-cis,cis-muconic acid, the
3-carboxymuconolactonizing enzyme or 4-carboxymuconolactonizing enzyme
may be disrupted.

[0021]Production of 3-carboxy-cis,cis-muconic acid according to the
invention is accomplished by introducing the gene-disrupted strain of the
invention into, for example, i) a host microorganism having a function of
metabolizing terephthalic acid to protocatechuic acid (terephthalate
assimilation), and lacking a terephthalate 2,3-ring cleavage function but
having a terephthalic acid 3,4-ring cleavage function or 4,5-ring
cleavage function, or ii) a host microorganism lacking terephthalate
assimilation but having "protocatechuate assimilation" by a
protocatechuate 2,3-ring cleavage function, 3,4-ring cleavage function or
4,5-ring cleavage function.

[0022]The invention will now be explained in detail using a gene-disrupted
strain with disrupted protocatechuate 4,5-ring cleavage function as an
example.

[0024]For the purpose of the invention, the protocatechuate 4,5-ring
cleavage enzyme gene group will be referred to as "pmd gene group." Of
the pmd gene group, the gene coding for the α-subunit of the enzyme
having dioxygenase activity that cleaves the protocatechuate 4,5-ring for
conversion to 4-carboxy-2-hydroxy-6-semialdehyde muconate will be
referred to as "pmdA1" (the amino acid sequence set forth in SEQ ID NO: 3
and the nucleotide sequence set forth in SEQ ID NO: 4), and the gene
coding for the β-subunit of the same will be referred to as "pmdB1"
(the amino acid sequence set forth in SEQ ID NO: 1 and the nucleotide
sequence set forth in SEQ ID NO: 2).

[0025]Also, the gene coding for the enzyme with dehydrogenase activity
that cleaves 4-carboxy-2-hydroxy-6-semialdehyde muconate for conversion
to 2H-pyran-2-one-4,6-dicarboxylic acid will be referred to as "pmdC".

[0026]The method of obtaining the gene coding for protocatechuate
4,5-ring-cleaving enzyme is not particularly restricted, and for example,
the gene may be obtained by preparing a suitable probe or primer based on
the data for the nucleotide sequence of the gene, and screening a cDNA
library or genomic DNA library for the strain, using the probe or primer.

[0027]The gene for protocatechuate 4,5-ring-cleaving enzyme may also be
obtained by PCR. The chromosomal DNA or cDNA library of the strain may be
used as template for PCR with a pair of primers designed so as to amplify
the nucleotide sequence of the gene. The PCR reaction conditions may be
set as appropriate, and for example, it may be carried out under
conditions in which reaction for 30 seconds at 94° C.
(denaturation), 30 seconds to 1 minute at 55° C. (annealing) and 2
minutes at 72° C. (extension) as one cycle, is carried out for 30
cycles followed by reaction for 7 minutes at 72° C. The amplified
DNA fragment may then be cloned in a suitable vector. The vector is
preferably selected as a vector that is autoreplicating in E. coli and
that is incapable of extrachromosomal autoreplication in Comamonas sp.

[0028]The procedures for preparation of the probe or primer, construction
of the cDNA library, screening of the cDNA library and cloning of the
target gene may be known to those skilled in the art, and for example,
they may be carried out according to the methods described in Molecular
Cloning: A laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., 1989 and Current Protocols in Molecular
Biology, Supplement pp. 1-38, John Wiley & Sons (1987-1997).

[0029](II) Construction of Gene-Disrupted Strain

[0030]The gene-disrupted strain of the invention is a disrupted strain
wherein protocatechuate 4,5-ring cleavage has been selectively disrupted.
More specifically, the gene coding for: (a) the amino acid sequence set
forth in SEQ ID NO: 1 or 3, or (b) the amino acid sequence set forth in
SEQ ID NO: 1 or 3 which has a deletion, substitution, addition and/or
insertion of one or more amino acids and exhibits protocatechuate
4,5-ring cleavage activity, present in the chromosomal DNA of microbial
cells, has been disrupted.

[0031]The gene may be a gene that comprises (a) the nucleotide sequence
set forth in SEQ ID NO: 2 or 4; or (b) a nucleotide sequence hybridizing
with DNA consisting of a nucleotide sequence complementary to the
nucleotide sequence of (a) under stringent conditions, and coding for an
enzyme with protocatechuate 4,5-ring cleavage activity.

[0032]Specifically, the gene to be disrupted may be the pmdB1 gene set
forth in SEQ ID NO: 2, the pmdA1 gene set forth in s SEQ ID NO: 4, or
both of these genes.

[0033]There are no particular restrictions on the range of "one or more"
in the phrase "amino acid sequence having a deletion, substitution,
addition and/or insertion of one or more amino acids" used throughout the
present specification, and it may be, for example, 1-20, preferably 1-10,
more preferably 1-7 and most preferably about 1-3.

[0034]The phrase "stringent hybridization conditions" used in the present
specification means "highly stringent conditions" under which a DNA chain
can hybridize to another DNA chain which is highly complementary to the
DNA chain, and which is designed so as to exclude significantly
mismatched DNA hybridization, or "moderately stringent conditions" under
which DNA double strands can form with a greater degree of base pair
mismatching than is possible under "highly stringent conditions". As
specific examples of "highly stringent conditions" there may be mentioned
0.015 M sodium chloride and 0.0015 M sodium citrate at 65-68° C.,
or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at
42° C. As specific examples of "moderately stringent conditions"
there may be mentioned 0.015 M sodium chloride and 0.0015 M sodium
citrate at 50-65° C., or 0.015 M sodium chloride, 0.0015 M sodium
citrate, and 20% formamide at 37-50° C.

[0035]As specific hybridizing DNA there may be mentioned DNA having at
least 60%, preferably at least 70%, more preferably at least 80%, even
more preferably at least 90%, yet more preferably at least 95% and most
preferably at least 97% homology with a polynucleotide containing the
nucleotide sequence set forth in SEQ ID NO: 2 or 4, as calculated using
analysis software such as BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA
[Methods in Enzymology, 183, 63 (1990)].

[0037]The gene-disrupted strain of the invention can be generated by
introducing a mutation and/or a selective marker into the gene coding for
protocatechuate 4,5-ring-cleaving enzyme obtained by PCR or cloning, to
obtain DNA lacking protocatechuate 4,5-ring cleavage activity, and then
using the DNA for homologous recombination.

[0038]The method for introducing a selective marker into the gene coding
for protocatechuate 4,5-ring-cleaving enzyme may be, for example, a
method in which the gene is cut with an appropriate restriction enzyme
and then an unrelated gene, preferably a selective marker gene that
allows selection of strains that have undergone homologous recombination,
is inserted. When no suitable restriction enzyme site is present, a
suitable restriction enzyme site may be introduced by PCR or the like. As
selective markers there are preferred drug resistance markers, and as
examples there may be mentioned kanamycin resistance genes, ampicillin
resistance genes and tetracycline resistance genes.

[0040]The DNA lacking protocatechuate 4,5-ring cleavage activity, used to
generate the gene-disrupted strain of the invention, may be any that
undergoes homologous recombination with a gene coding for the
corresponding protocatechuate 4,5-ring-cleaving enzyme on the chromosome
under physiological conditions, i.e. in microbial cells, having
sufficient homology to thus allow disruption of the protocatechuate
4,5-ring-cleaving enzyme gene. The homology is preferably 80% or greater,
more preferably 90% or greater and most preferably 95% or greater. The
DNA used for homologous recombination may also be a portion of the
protocatechuate 4,5-ring-cleaving enzyme gene, so long as it undergoes
homologous recombination with a gene coding for the corresponding
protocatechuate 4,5-ring-cleaving enzyme on the chromosome under
physiological conditions, i.e. in microbial cells, thereby allowing
disruption of the protocatechuate 4,5-ring-cleaving enzyme gene. The
portion referred to here may be a length of preferably 50 or more
nucleotides, and more preferably 100 or more nucleotides.

[0041]In order to generate a gene-disrupted strain by homologous
recombination, first there is constructed DNA containing the nucleotide
sequence of the protocatechuate 4,5-ring-cleaving enzyme gene or
recombinant DNA comprising the mutated DNA having an appropriate
selective marker inserted therein. When a drug resistance marker is used
as the selective marker, it is necessary to select a resistance gene for
a drug to which the wild type strain prior to homologous recombination is
sensitive. This will allow discernment between strains that have
undergone homologous recombination and strains that have not, based on
growth in the presence of an antibiotic. Next the DNA having the
selective marker inserted into the gene sequence is introduced into the
strain by electroporation or the like, and then selection is carried out
using the marker to incorporate the target gene into the host
microorganism chromosomes by homologous recombination.

[0042]The parent strain of the protocatechuate 4,5-ring-cleaving enzyme
gene-disrupted strain is not particularly restricted so long as it is a
soil bacterium of Comamonas, Pseudomonas, Bacillus, Lactobacillus,
Streptococcus, Saccharomyces, Candida or the like. The parent strain may
be a strain derived from a microorganism that is capable of
"terephthalate assimilation", or a strain derived from a microorganism
that is incapable of "terephthalate assimilation" but is capable of
"protocatechuate assimilation".

[0043]A microorganism that is capable of terephthalate assimilation is
able to metabolize terephthalic acid to protocatechuic acid. As examples
of strains capable of terephthalate assimilation, there may be mentioned
Comamonas sp. E6, Pseudomonas putida PPY1100, Comamonas testosteroni (C.
testosteroni) T-2, C. testosteroni YZW-D, Derftia tsuruhatensis T7,
Rhodococcus sp. DK17 and Rhodococcus jostii RHA1. Of these, Comamonas sp.
E6 is a strain that retains ability to metabolize terephthalic acid to
protocatechuic acid and ability to metabolize protocatechuic acid to
2H-pyran-2-one-4,6-dicarboxylic acid (4,5-ring cleavage function), but
does not retain 2,3-ring cleavage function and 3,4-ring cleavage
function. When Comamonas sp. E6 was used as the parent strain, the
production efficiency for 3-carboxy-cis,cis-muconic acid was roughly
equivalent to a fermentative production system from vanillic acid to
2H-pyran-2-one-4,6-dicarboxylic acid (Japanese Unexamined Patent
Publication No. 2005-278549) or a fermentative production system to
3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone (Japanese
Patent Application No. 200-218524).

[0044]A microorganism that is not capable of terephthalate assimilation
but is capable of protocatechuate assimilation has a 2,3-ring cleavage
function, 3,4-ring cleavage function or 4,5-ring cleavage function. As
examples of such microorganisms there may be mentioned microorganisms
belonging to the genus Pseudomonas, Bacillus, Burkholderia and
Agrobacterium.

[0045]A gene-disrupted strain with disrupted protocatechuate 2,3-ring
cleavage function or 3,4-ring cleavage function can also be generated in
the same manner as the aforementioned 4,5-ring cleavage
function-disrupted strain.

[0046]A microorganism capable of terephthalate assimilation will generally
have more developed terephthalate uptake and metabolism compared to a
microorganism incapable of terephthalate assimilation, and higher
conversion efficiency would be expected.

[0047]Incidentally, 3-carboxy-cis,cis-muconic acid can also be efficiently
produced using a strain that is not a gene-deleted strain according to
the invention but which retains protocatechuate 3,4-ring cleavage
function and does not further catabolize the 3-carboxy-cis,cis-muconic
acid cleavage product. Pseudomonas putida PPY1100 may be mentioned as an
example of such a strain, but the reaction efficiency is slightly lower
with Pseudomonas putida PPY1100 compared to Comamonas sp. E6.

[0048]For even more efficient production of 3-carboxy-cis,cis-muconic
acid, it is more preferred to use a gene-disrupted strain having
disrupted protocatechuate 2,3- and 4,5-ring cleavage activity, and also
having disrupted 3-carboxy-cis,cis-muconic acid metabolism activity in
order to completely eliminate the possibility of further catabolism of
3-carboxy-cis,cis-muconic acid.

[0050]The plasmid is a plasmid comprising genes for enzymes that catalyze
a multistage process for production of 3-carboxy-cis,cis-muconic acid
from terephthalic acid via protocatechuic acid (positive regulator,
TPA-transporter, TPA-DOX, DCD-dehydrogenase, NADPH-reductase,
protocatechuate 3,4-ring cleavage gene), in that order from the upstream
end (FIG. 8).

[0051]The positive regulator gene (tphR) is the DNA molecule set forth in
SEQ ID NO: 11 in Japanese Patent Application No. 2005-298242 (SEQ ID NO:
9 in the present specification), the TPA-transporter gene (tphC) is the
DNA molecule set forth in SEQ ID NO: 13 in the same specification (SEQ ID
NO: 10 in the present specification), the TPA-DOX genes (tphA2, tphA3)
are the DNA molecules set forth in SEQ ID NO: 2 and 4 in the same
specification (SEQ ID NO: 11 and 12, respectively, in the present
specification), the DCD-dehydrogenase gene (tphB) is the DNA molecule set
forth in SEQ ID NO: 6 in the same specification (SEQ ID NO: 13 in the
present specification), and the NADPH-reductase gene (tphA1) is the DNA
molecule set forth in SEQ ID NO: 8 in the same specification (SEQ ID NO:
14 in the present specification). The protocatechuate 3,4-ring cleavage
genes (pcaH and G) used for the invention are DNA fragments obtained from
Pseudomonas putida KT2440, and the nucleotide sequence of the PcaH gene
is set forth in SEQ ID NO: 1 of Japanese Patent Application No.
2006-218524 while the nucleotide sequence of the PcaG gene is set forth
in SEQ ID NO: 3 of the same specification (SEQ ID NO: 15 and 16 of the
present specification). In the present specification, tphR, tphC, tphA2,
tphA3, tphB and tphA1 will also be collectively referred to as "Tph gene
cluster" or "Tph gene group".

[0052]Specifically, the plasmid for fermentative production of
3-carboxy-cis,cis-muconic acid from terephthalic acid according to the
invention may be constructed as illustrated in FIGS. 6 to 8.

[0053](1) First, the PcaH gene (SEQ ID NO: 1) and PcaG gene (SEQ ID NO: 2)
listed in Japanese Patent Application No. 2006-218524 are ligated to a
multicloning site in the gene coding for the α-fragment of LacZ,
present downstream from the pBluescript LacZ promoter, using a known
ligase, to construct recombinant plasmid pBluescript II SK.sup.-/pcaHG.

[0054]Next, a known ligase is used to ligate a DNA fragment obtained by
end treatment after cutting pBluescript II SK.sup.-/pcaHG with
restriction enzymes PvuII and BamHI, with a DNA fragment obtained by end
treatment after cutting an Amp promoter-containing plasmid with
restriction enzyme XbaI, to construct recombinant plasmid pKHG (FIG. 6).

[0055](2) Next, a known ligase is used to ligate a DNA fragment obtained
by end treatment after cutting a chloramphenicol resistance
gene-containing plasmid with restriction enzyme Cfr13I, with a DNA
fragment obtained by end treatment after cutting pKHG with restriction
enzyme KpnI, to construct recombinant plasmid pKHG/C (FIG. 7).

[0056](3) Also, a known ligase may be used to ligate a DNA fragment
obtained by cutting recombinant plasmid pHE96/pBluescript II SK (+),
having tphR, tphC, tphA2, tphA3, tphB and tphA1 ligated in that order
from the upstream end and shown in FIG. 2 of Japanese Patent Application
No. 2005-298242, with restriction enzyme EcoRI, with a DNA fragment
obtained by end treatment after cutting the pKHG/C obtained in (2) with
EcoRI, to construct recombinant plasmid pKTphHG/C.

[0057]A known method such as a protoplast method, competent cell method or
electroporation method may be used for transformation of the
gene-disrupted strain of (I) using the recombinant plasmid pKTphHG/C.

[0058]Selection of transformants may be accomplished based on a selective
marker for the plasmid used, such as drug resistance acquired by DNA
recombination in the transformants. The transformants containing the
recombinant plasmid of interest are preferably selected from among the
transformants by colony hybridization using a partial DNA fragment of the
gene as the probe. Labeling of the probe may be carried out using a
radioactive isotope, digoxigenin, an enzyme or the like.

[0059](IV) The Fermentative Production of 3-Carboxy-Cis,Cis-Muconic Acid
and/or 3-Carboxymuconolactone

[0060]The transformants of the invention of (II) may be cultured under
appropriate conditions in the presence of terephthalic acid, using medium
containing a carbon source, nitrogen source, metal salts, minerals,
vitamins and the like. The pH of the medium may be a pH in a range that
allows growth of the transformants, and the pH is preferably adjusted to
about 6-8. The culturing conditions may be shake culturing or submerged
culturing for 2-7 days at 15-40° C. and preferably 28-37°
C.

[0061]Ordinary isolation and purification methods for organic compounds
may be used for isolation and purification of 3-carboxy-cis,cis-muconic
acid from the cultured gene-disrupted strain. For example, upon
completion of culturing, the cells are collected by centrifugal
separation and suspended in aqueous buffer, and then disrupted using an
ultrasonic disruptor or the like to obtain a cell-free extract. The
target substance may be obtained by ordinary isolation and purification
methods for organic compounds, from the supernatant obtained by
centrifugal separation of the cell-free extract.

[0062]Acid treatment of the 3-carboxy-cis,cis-3-muconic acid obtained in
this manner, or the culture solution containing the unpurified
3-carboxy-cis,cis-3-muconic acid, will allow conversion to
3-carboxymuconolactone at a high yield. The acid used is preferably
hydrochloric acid at about pH 1-2.

[0063]The 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone
obtained by the production process of the invention, as a plastic
material, chemical product material or the like, can exhibit functions
different from 2H-pyran-2-one-4,6-dicarboxylic acid or higher functions
thereof, and therefore can serve as a useful plastic material.

EXAMPLES

[0064]The present invention will now be described in greater detail by
examples, with the understanding that the invention is not limited to
these examples.

Example 1

Cloning of pmd Gene Group

(1) Amplification of Protocatechuate 4,5-Dioxygenase Gene by PCR and
Sequencing

[0065]A region of high homology was identified from alignment of the amino
acid sequences deduced from the ligA gene of Sphingomonas paucimobilis
SYK-6, the pcmA gene of Arthrobacter keyseri 12B, the pmdA1B1 gene of
Comamonas testosteroni BR6020 and the fldVU gene of Sphingomonas sp.
LB126, and PCR was conducted using the following αF/βR primer,
with the total DNA of the Comamonas sp. E6 protocatechuate
4,5-dioxygenase gene as template.

[0066]As a result, amplification was seen at the predicted size of 900 bp.
The PCR product was then used as template for PCR between
βF-βR, using the nested primer βF. As a result,
amplification was seen of a 450 by fragment of the predicted size. The
obtained PCR product was subcloned in pT7Blue vector to determine the
nucleotide sequence of the 449 by fragment. A homologous sequence search
was conducted in the DDBJ database, and the gene coding for the Comamonas
testosteroni BR6020 protocatechuate 4,5-dioxygenase β-subunit (DDBJ
Accession No.: AF305325) showed 99% homology on the amino acid level.
Also, homology of 68% was found with Sphingomonas paucimobilis SYK-6 ligB
(DDBJ Accession No.: AB035122), and 66% with both Arthrobacter keyseri
12B pcmA (DDBJ Accession No.: AF331043) and Sphingomonas sp. LB126 fldU
(DDBJ Accession No.: AJ277295).

[0067]Positive clones were obtained from a cosmid library containing E6
SalI partial digestion fragments, by colony hybridization using the PCR
product as the probe. The cosmid extracted from the obtained positive
clones was digested with SalI and subjected to Southern hybridization
with the same probe, which produced hybridization of 10 kb, and the
cosmid was named pPV10.

[0068]Clone pKS10F(R) was constructed by reciprocal bidirectional
insertion of the pPV10 10-kb SalI fragment downstream from the
pBluescript II KS (+) lac promoter. Three fragments of 1.9 kb, 3.2 kb and
4.9 kb, obtained by digestion of pKS10F with SalI and XhoI, were blunted
and inserted into the SmaI site of pBluescript II KS(+). After reciprocal
bidirectional insertion of these fragments downstream from the lac
promoter, the obtained plasmids were named pK1SXF(R), pK3XF(R) and
pK4XSF(R), respectively (FIG. 1). The full nucleotide sequences of the
pK1SXF(R) 1.9-kb SalI-XhoI fragment and the 3.2-kb XhoI fragment in pK3X
were determined. Sequencing of the pKSTS 2.2-kb StuI fragment also
confirmed that these fragments are adjacent across XhoI (FIG. 1). In FIG.
1, Plac represents the lac promoter, with the arrow indicating the
direction of transcription.

(4) Isolation of Region Upstream from 10-Kb SalI Fragment

[0069]In order to isolate the region upstream from the pPV10 10-kb SalI
fragment, a library was prepared by cloning of a Sad digest of E6 total
DNA in charomid 9-36. A 1,150 by fragment obtained by digestion of the
1.9-kb SalI-XhoI fragment with SalI and EcoRI was used for colony
hybridization, to isolate pCS18 having a 10-kb SacI fragment containing
pmdA1B1 (FIG. 2). After nucleotide sequencing to confirm that the pSC18
2.1-kb SacII fragment containing pmdA1B1 is adjacent to the 6.1-kb
SacI-SalI fragment, clone pSA2-6F(R) obtained by blunting the 6.1-kb
SacI-SalI fragment and reciprocal bidirectional insertion at the EcoRV
site downstream from the pBluescript II KS(+) lac promoter was generated,
and the full nucleotide sequence was determined (FIG. 3). SEQ ID NO: 8
includes the gene sequence comprising the genes pmdJ (base numbers
1-1029), pmdK (base numbers 1162-1845), pmdI (base numbers 1942-2934),
pmdA1 (SEQ ID NO: 2), pmdB1 (SEQ ID NO: 4 and pmdC (base numbers
4427-5386), shown in FIG. 3.

Example 2

Construction of pmdB1 Gene-Disrupted Strain

[0070](1) Preparation of pmdB1 Gene-Disrupting Plasmid

[0071]A 3.6-kb SalI-EcoRV fragment containing the pmdA1B1C gene was cut
out from pKS10F and inserted into a SalI-EcoRV digest of pBluescript II
KS(+) to obtain pSDB36. It was then digested with StuI within the pmdB1
gene, and a 1.2-kb EcoRV fragment containing the pIK03-derived kanamycin
resistance gene was inserted therein (the underlined portion of the
sequence of SEQ ID NO: 1). Insertion of the fragment in the same
direction as the pBluescript II KS(+) lac promoter transcription
direction was confirmed by SmaI digestion, thus obtaining pKS78F. A
4.9-kb SalI-EcoRV fragment was cut out from pKS78F and inserted at the
SalI-SmaI site of pK19 mobsacB to construct plasmid pDBKM for generated
of a pmdB1-disrupted strain (FIG. 4).

(2) Construction of pmdB1 Gene-Disrupted Strain

[0072]E6 precultured in 3 ml of LB medium was inoculated at 1% into 10 ml
of LB medium, for main culturing. After growing at OD600=0.5, the culture
was centrifuged at 5,000 rpm, 4° C., 15 minutes and the cells were
collected. After rinsing twice in 1 ml of 0.3 M sucrose, it was suspended
in 1 ml of 0.5 M sucrose. Next, 1 μl of the gene-disrupting plasmid
pDBKM prepared to 1 μg/μl was added to 100 μl of the culture and
subjected to pulsing using a Gene pulser (Bio-Rad), under conditions with
a resistance of 800Ω, a voltage of 12 V and an electrostatic
capacity of 25 μF. Immediately after pulsing, 1 ml of LB medium was
added and culturing was carried out at 30° C. for 6 hours. A 300
μl portion thereof was coated onto 100 μg/ml kanamycin-containing
LB medium.

[0073]The obtained kanamycin-resistant strains were collected with 1 ml of
LB medium, seeded in 10 ml of LB medium containing 10% sucrose, and
cultured for 12 hours. A 200 μl portion was then transferred into
fresh LB medium and the procedure was repeated three times. Finally, the
culture solution was coated onto 100 μg/ml kanamycin-containing LB
medium and the obtained colonies were used as candidate gene-disrupted
strains.

(3) Confirmation of pmdB1 Gene Disruption

[0074]The candidate gene-disrupted strains were precultured in 3 ml of 100
μg/ml kanamycin-containing LB medium, and inoculated at 1% into 10 ml
of culture medium for main culturing. Next, the total DNA of the
candidate gene-disrupted strains were recovered and digested with EcoRV,
after which a 3.4-kb HindIII-KpnI fragment containing pmdA1B1 and a
1.2-kb EcoRV fragment containing the pIK03-derived kanamycin resistance
gene were used as probes for Southern hybridization (FIG. 5). Lanes 1 and
3 represent the total DNA of the wild type E6 digested with EcoRV, and
lanes 2 and 4 represent the total DNA of the gene-disrupted strain
digested with EcoRV. The probe used was a pmdB1-containing 3,4-kb
HindIII-KpnI fragment for lanes 1 and 2 and a kanamycin (Km) resistance
gene-containing 1.2-kb EcoRV fragment for lanes 3 and 4.

[0079]2-1) Recombinant plasmid pKTphHG/C was used to transform E. coli
HB101, and the transformants were shake cultured at 37° C. for 18
hours in LB medium (100 ml) containing 25 mg/L kanamycin, after which the
recombinant plasmid pKTphHG/C was extracted from the proliferated
cultured cells.

[0080]2-2) The gene-disrupted strain generated in Example 2 (Comamonas sp.
EDB) was cultured at 28° C. for 23 hours in 500 ml of LB liquid
medium and cooled on ice for 30 minutes. The cells were collected by
centrifugation at 4° C., 10,000 rpm for 10 minutes, and after mild
rinsing with 500 ml of 0° C. distilled water, they were
re-centrifuged. This was followed by additional mild rinsing with 250 ml
of 0° C. distilled water and re-centrifugation. This was further
followed by additional mild rinsing with 125 ml of 0° C. distilled
water and re-centrifugation. The collected microbial cells were suspended
in distilled water containing 10% glycerol and stored at 0° C.

[0081]2-3) After placing 4 μl of distilled water containing about 0.05
μg of the DNA of plasmid pKTphHG/C of (1) in a 0.2 cm cuvette, 40
μl of the cell solution suspended in distilled water containing 10%
glycerol obtained in 2-2) above was added, and the mixture was subjected
to electroporation under conditions of 25 μF, 2500 V, 12 msec.

[0082]2-4) The total amount of treated cells was seeded in 10 ml of LB
liquid medium and cultured at 28° C. for 6 hours. After culturing,
the cells were collected by centrifugation, developed on an LB plate
containing 25 mg/L kanamycin, 50 mg/L ampicillin and 30 mg/L
chloramphenicol and cultured at 28° C. for 48 hours, to obtain
transformants retaining plasmid pKTphHG/C and exhibiting chloramphenicol
resistance. The cells were designated as strain pKTphHG/C/Comamonas sp.
ECB.

[0084]2-6) When the OD518 reached 3 with culturing using a 10 L-volume jar
fermenter, 500 ml of culture solution was removed from the fermenter into
an Erlenmeyer flask and stored on ice.

[0085]2-7) To the culture solution in the fermenter that had reached OD518
of 3 there was added 42 g of terephthalic acid dissolved in 500 ml of a
0.1 N NaOH aqueous solution (adjusted to pH 8.5), over a period of 10-12
hours using a peristaltic pump. In order to prevent reduction in the pH
of the culture solution with production of 3-carboxy-cis,cis-muconic acid
as the reaction proceeded, a 0.1 N NaOH solution was added with a
peristaltic pump connected to a pH sensor to maintain the pH of the
culture solution. Progress of the reaction was confirmed by HPLC. After
36 hours, the added terephthalic acid had virtually disappeared. A 500 ml
portion of the ice-cooled cell suspension prepared in 2-5) was added to
the culture solution in the fermenter and culturing was continued for 12
hours.

[0086]2-8) Upon completion of the reaction, the medium in the fermenter
was transferred to a plastic container (bucket). The cell component was
precipitated and removed from the culture solution by centrifugal
separation (6000 rpm, 20° C.), hydrochloric acid was added to the
obtained supernatant to lower the pH to below 1.0, and the mixture was
stored at low temperature for conversion of the 3-carboxy-cis,cis-muconic
acid to 3-carboxymuconolactone. After confirming complete conversion to
3-carboxymuconolactone by GC-MS, an organic solvent (ethyl acetate) was
used for extraction of the 3-carboxymuconolactone. The amount of
extracted and dried 3-carboxymuconolactone reached approximately 9.8 g
from 1 L of culture solution, which was a yield of about 87% as the ratio
of added substrate (terephthalic acid). The obtained
3-carboxymuconolactone was further treated with active carbon and the
structure was confirmed by its NMR and MS spectra.